Glycogen synthase kinase 3 (GSK-3) is a serine and threonine kinase that regulates a plethora of physiological functions in the periphery and central nervous system ranging from differentiation and development, to metabolism, cell cycle regulation, and neuroprotection (see e.g., Woodgett et al., EMBO J. 1990, 9:2431; Woodgett et al., In Glycogen Synthase Kinase 3 (GSK-3) and Its Inhibitors: Drug Discovery and Development; John Wiley and Sons, Inc.: Hoboken, N.J., 2006, pg 3; Jope et al., Trends in Biochemical Sciences, 2004, 29:95; Meijer et al., Trends in Pharmaceutical Sciences, 2004, 25:471; Lei et al., International Journal of Alzheimer's Disease, 2011, 2011:9; and Thorne et al., Nat. Chem. Biol. 2015, 11:58). Two paralogues, GSK-3α (51 kDa) and GSK-3β (47 kDa) are known and there is high homology in their kinase domains (ca. 98% identical in their catalytic domains) (see e.g., Woodgett et al., Methods Enzymol, 1991, 200:564; and Harr et al., Nat. Struct. Mol. Bio. 2001, 8:593). GSK-3 plays a significant role in several pathologies including Alzheimer's disease (AD), bipolar disorder, schizophrenia, Huntington's disease, type-II diabetes, stroke, cardiac ischemia, age-related loss of bone and muscle, chronic inflammatory conditions, and in some cancers (see e.g., Martinez et al., Current Topics in Medicinal Chemistry, 2013, 13:1808).
Specifically for neurodegenerative diseases, molecular imaging of GSK-3 can indicate target engagement by GSK-3 therapeutics and offer a path to diagnostic agents that not only correlates with early cognitive impairment, but also increased tau hyperphosphorylation, (see e.g., Martinez et al., Current Topics in Medicinal Chemistry, 2013, 13:1808; Takahashi et al., Journal of Neurochemistry, 1994, 63:245); and Wang et al., Nat. Rev. Neurosci. 2016, 17:22) increased amyloid-β production (see e.g., DaRocha-Souto et al., Neurobiol. Dis. 2012, 45:425) and local plaque-associated glial-mediated inflammatory responses; all of which are hallmarks of AD and non-AD tauopathies. This has led to the GSK-3 Hypothesis of Alzheimer's Disease (see e.g., Hooper et al., J. Neurochem, 2008, 104:1433). GSK-3 plays a key role in AD evident from: (i) the abundance and dysregulation of GSK-3 in the AD brain (see e.g., Lucas et al., Embo. J. 2001, 20:27; Hernandez et al., J. Neurochem, 2002, 83:1529; Fuster-Matanzo et al., PLoS ONE, 2011, 6:e27262; Sirerol-Piquer et al., Hippocampus, 2011, 21:910; and Leroy et al., Neuropathology and Applied Neurobiology, 2007, 33:43), (ii) reduced tau phosphorylation (pTau) induced by treatment with GSK-313 inhibitors (see e.g., Hurtado et al., J. Neurosci, 2012, 32:7392) and, (iii) genetic studies suggesting GSK-3 is fundamental in the pathogenesis of sporadic and familial AD. The defining neuropathological lesions of AD are amyloid-β (Aβ) senile plaques and tau neurofibrillary tangles, both of which appear many years before the onset of symptoms of cognitive impairment (see e.g., Hurtado et al., J. Neurosci, 2012, 32:7392; and Llorens-Maritin et al., Frontiers in Molecular Neuroscience, 2014, 7:46). GSK-313 has been recognized as one of the principal kinases involved in phosphorylation of tau, an essential protein for stabilization of intracellular microtubules. The phenotypic forms of neurofibrillary tangles with fine structure composed of paired helical fragments are typically a result of tau aggregation induced by hyperphosphorylation of tau. Over-expression of GSK-3 has been studied extensively in transgenic animal models of AD and in AD patients, whose hippocampal brain regions have elevated GSK-3β expression and/or enzymatic activity (see e.g., Hurtado et al.). The role of GSK-3β in tau phosphorylation makes it a particularly attractive therapeutic target for AD and non-AD tauopathies (see e.g., Llorens-Maritin et al.).
The pertinence of GSK-3 in diverse diseases has led to long-standing, world-wide efforts by major pharmaceutical companies to develop small molecule inhibitors as therapeutics for this target (see e.g., Cohen et al., Nat. Rev. Drug Discov. 2004, 3:479). Clinical translation of potent GSK-3 therapeutics for neurodegenerative disease have faced three major hurdles: 1) poor GSK-3 selectivity over other central nervous system (CNS) targets and closely related kinases; 2) low blood-brain barrier (BBB) penetration and 3) chronic toxicity. A PET radiotracer for GSK-3 could aid the many ongoing clinical research efforts to develop GSK-3 targeted therapeutics by indicating the success and extent of engagement by GSK-3 inhibitors in the brain.
Development of a robust and reliable radiotracer for detecting biomarkers for dementias is among the most sought-after goals in nuclear medicine. Current clinical neuroimaging agents for AD are generally classified as: (i) enzyme trapped substrates (e.g., [18F]FDG); Bohnen et al., J. Nucl. Med. 2012, 53:59) (ii) amyloid plaque or tau protein targeted tracers (e.g., [18F]Amyvid, [11C]PiB, [18F]T807; Okamura et al., IDrugs, 2010, 13:890; Rowe et al., J. Nucl. Med. Technol. 2013, 41:11; Marquie et al., Ann. Neurol. 2015, 78, 787), (iii) neuroreceptor imaging probes (e.g., [18F]FPEB, [18F]GE179; Stephenson et al., J. Nucl. Med., 2015, 56:489; and McGinnity et al. J. Nucl. Med. 2014, 55:423). While the advent of imaging and fluid biomarkers of brain β-amyloidosis has propelled the field forward and improved the ascertainment of early stages of AD, the levels of deposited Aβ do not relate robustly to the clinical phenotype. From a PET imaging perspective, despite substantial progress in AD biomarker development over the past decade, we do not yet have a radiotracer that combines two key properties: 1) positivity at an early stage of disease; and 2) good correlation with progression of symptoms and signs of the disease in AD and non-AD tauopathies.
Similar to the challenges for advancing a GSK-3 therapeutic for the CNS, the greatest obstacles for molecular neuroimaging of GSK-3 has been the lack of potent and highly selective small molecules with reasonable brain penetration that are capable of being radiolabelled. Our initial work on this target as a PET radiotracer for GSK-3 (see e.g., Vasdev et al., Bioorganic & Medicinal Chemistry Letters, 2005, 15:5270; and Hicks et al., Molecules, 2010, 15:8260) focused on the synthesis of 11C-labelled isotopologues of AR-A014418 (see e.g., Vasdev et al.; and Hicks et al., Bioorganic & Medicinal Chemistry Letters, 2012, 22:2099), and subsequently other laboratories explored different scaffolds. Only three other radiotracers for GSK-3 have been studied in vivo. [11C]SB-216763 showed good brain uptake in rodents and non-human primates (NHPs) but was not selective against other structurally similar kinases (see e.g., Wang et al., Bioorganic & Medicinal Chemistry Letters, 2011, 21:245; and Li et al., ACS Medicinal Chemistry Letters, 2015, 6:548). [11C]PyrATP-134 and 11C-oxadiazole35 based radiotracers failed to show appreciable uptake in vivo (see e.g., Cole et al., Nuclear Medicine and Biology, 2014, 41:507; and Kumata et al., Bioorganic & Medicinal Chemistry Letters, 2015, 25:3230). None of the PET radiotracers for GSK-3 have yet proven to be successful for in vivo imaging studies with specificity and/or suitable brain uptake. Thus a striking need for PET neuroimaging of GSK-3 remains, specifically for clinical research applications in AD and non-AD tauopathies (see e.g., Holland et al., Journal of Labelled Compounds and Radiopharmaceuticals, 2014, 57:323).